According to CDC data, aside from non-melanoma skin cancer, breast cancer is the most common form of cancer in women. In fact, breast cancer is the number one cause of cancer death in Hispanic women, and it is also the second most common cause of cancer death across various ethnic groups in white, black, Asian/Pacific Islander, and American Indian/Alaska Native women. In 2004 (the most recent year numbers are available), 186,772 women and 1,815 men were diagnosed with breast cancer. The most common type of breast cancer, called ductal cancer, occurs in the cells of the breast ducts. Cancer that begins in the lobes or lobules is called lobular breast cancer. Lobular cancer is more often found in both breasts than other types of breast cancer. A third type of breast cancer, inflammatory breast cancer, is less common than the other two types.
The causative factors are not completely understood but are thought to be composed of both hereditary (e.g., BRCA1 and BRCA2 genes) and environmental (e.g., high fat diets, obesity, and smoking) factors. Despite multiple causative factors of breast cancer, the survival and growth of the breast cancer cells largely depend upon the common set of molecular signaling pathways in the cells.
Several molecular targets mediating interactive signal pathways have been implicated in the breast cancer formation and spread. Among these targets, EGFR, Raf-1 and mTOR are three prominent oncogenes in breast cancer promotion. Small molecule drugs and monoclonal antibodies have been developed or designed to block these targets with varying degrees of success. We believe that inhibition of these targets at the messenger RNA (mRNA) level will have a greater impact on the inhibition of breast cancer growth.
The Role of Epidermal Growth Factor Receptor in Breast Cancer
The epidermal growth factor receptor (EGFR) is the product of an activated oncogene. Elevated expression of EGFR is frequently detected in a wide variety of epithelial cell carcinomas, including breast, lung, head and neck, and cervical cancers, and it has been correlated with a poor prognosis. Agents that target members of the EGFR family have been approved for cancer therapy, such as ZD1839/Iressa and Herceptin.
The family of seven EGFR ligands includes EGF, TGFα, heparin-binding EGF-like growth factor (HB-EGF), amphiregulin (AR), betacellulin (BTC), epiregulin (EPR), and epigen. All of these ligands are synthesized as type-I transmembrane pro-ligands that consist of one or more EGF-like domains in their extracellular segments that are proteolytically cleaved to yield mature growth factors.
The EGFR signaling can also be transactivated by ligands of G-protein coupled receptors (GPCR). It was demonstrated that EGFRs were rapidly phosphorylated on the tyrosine residues after stimulation with the GPCR agonists, such as endothelin-1, lysophosphatidic acid or thrombin. The GPCR-EGFR cross-talk mechanism is now considered to be a widely interactive signal towards the activation of the MAP kinase pathway. EGFR transactivation is achieved by the action of many GPCRs and a variety of G-proteins.
The Gastrin-releasing peptide (GRP) binding to its receptor of GRPR has been shown to contribute to carcinogenesis by autocrine growth stimulation, resulting in acceleration of tumor cell proliferation. The importance of this pathway is underscored by the involvement of both autocrine growth pathways, TGF/EGFR and GRP/GRPR, in various types of cancers, including head and neck, lung, prostate, mammary, pancreas, colon, and ovary.
The Role of Raf-1 kinase in Breast Cancer
The Raf-1 kinase, a 72-kDa cytoplasmic serine-threonine kinase, plays a central role as a second messenger in signal transduction. After ligand binding to a variety of transmembrane tyrosine kinase growth factor receptors, including epidermal growth factor (EGF) receptor, the 72-kDa Raf-1 kinase is activated through phosphorylation by other cellular kinases to become a 74-kDa phosphoprotein.
The Raf-1 kinase is constitutively activated in many transformed cells either directly, by mutations within its amino-terminus regulatory region, or indirectly, due to overstimulation by autocrine growth factors or activated proximal oncogenes. Several human breast cancer cell lines have been reported to express varied amounts of EGF receptor to influence the level of Raf-1 protein expression as well as the proportion of Raf-1 expressed in the higher molecular weight form. Effects of serum starvation and stimulation with EGF on the Raf-1 protein were studied in T47D, BT474, and MDA-MB231 cells by immuno-precipitation. Raf-1 protein was pulled down from cell lysates with an anti-Raf-1 antibody for immunoblot analysis. [3H]Thymidine incorporation by these cells after EGF stimulation was also used as a measure of DNA synthesis, an indicator of cell cycle progression. In all three breast cancer cell lines studied, the Raf-1 protein was identified in a combination of 70-kDa and a 74-kDa forms. The level of Raf-1 was similar in all the three cell lines and appeared to be unrelated to EGF receptor expression on the cell surface. The majority of the protein was found in the 74-kDa form even after serum starvation. A minor shift from lower to higher molecular weight form of Raf-1 was apparent in cells treated with EGF, and increased [3H] thymidine incorporation could be demonstrated in two of the cell lines after EGF stimulation. Baseline expression of the 74-kDa or activated form of the Raf-1 kinase appeared to be elevated in the breast cancer cells studied, indicating constitutive activation.
Breast cancer disease progression may be characterized by a switch from hormone-dependent to hormone-independent growth that involves several cellular alterations and is a major problem in the treatment of breast cancer. Expression of a constitutively activated Raf in ER+ MCF-7 human breast cancer cells results in estrogen-independent growth, suggesting that activation of growth factor signaling pathways through Raf may confer a selective advantage for growth of breast cancer cells under estrogen-deprived conditions.
Raf-1 kinase inhibitor protein (RKIP) was originally identified as the first physiologic inhibitor of the Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) pathway. This pathway regulates fundamental cellular functions, including those that are subverted in cancer cells, such as proliferation, transformation, survival, and metastasis. Recently, RKIP has been recognized as a strong candidate for a metastasis suppressor gene in cell and animal model systems. Clinical studies have indicated that that in human breast cancer, RKIP is a metastasis suppressor gene whose expression must be down-regulated for metastases to develop. RKIP expression is independent of other markers for breast cancer progression and prognosis. Therefore, inhibition of Raf-1 may contribute to control the metastases of breast cancer as well.
The Role of mTOR in Breast Cancer
The mammalian target of rapamycin (mTOR) is a serine-threonine kinase member of the phosphatidylinositol 3-kinase (PI3K) pathway, which is involved in multiple biologic functions, including transcriptional and translational control of downstream members' activities. mTOR is a downstream mediator in the PI3K/Akt signaling pathway and plays a critical role in cell survival. In breast cancer, this pathway can be activated by various membrane receptors, including the HER (or ErbB) family of growth factor receptors, the insulin-like growth factor receptor, and the estrogen receptor. There is evidence suggesting that Akt promotes breast cancer cell survival and resistance to chemotherapy, such as trastuzumab and tamoxifen.
Rapamycin is a specific mTOR antagonist that targets this pathway and blocks the downstream signaling elements, resulting in cell cycle arrest in the G1 phase. Targeting the Akt/PI3K pathway with mTOR antagonists may increase the therapeutic efficacy of breast cancer therapy.
Treatment of Breast Cancer
There are different types of treatment for patients with breast cancers. Four major methods of standard treatment of breast cancers are surgery, radiation therapy, chemotherapy, and hormone therapy. New types of treatment are also being tested in clinical trials now. These include the following: sentinel lymph node biopsy followed by surgery, high-dose chemotherapy with stem cell transplant, monoclonal antibodies as adjuvant therapy, and tyrosine kinase inhibitors as adjuvant therapy.
When discovered early, breast cancer can be treated with the above-mentioned therapies with a high rate of survival. The 5-year survival rate is now reaching over 70%, and the 10-year survival rate is about 50%. However, once the cancer spreads out to other tissues or organs, the survival rate with any of the treatments falls dramatically. Treatment of breast cancer depends on the types of cancers with varying degrees of success. Despite the recent integration of more powerful endocrine agents into breast cancer care, resistance to all forms of endocrine therapy remains a major problem. Therefore, more effective new therapies are absolutely needed.
An attractive approach for therapeutic intervention would be to inhibit all the targets along the pathways of EGFR, Raf-1 and mTOR, which all contribute to breast cancer growth. The three genes have been shown individually to be involved in the development of breast cancer. We hypothesized that a new, more effective therapeutic approach would be to suppress all three oncogenes simultaneously and preferably in combination with different anti-cancer agents.
Gene Inhibition by siRNA as an Alternative Therapeutic
Major advances in molecular biology, cellular biology and genomics have greatly enhanced our knowledge about gene regulation mechanisms in cancers. Gene silencing methods, such as antisense and ribozymes, have been shown to down-regulate disease related genes. Novel technologies, such as siRNA, have been developed and tested to show the safety and effectiveness of disease treatment, including but not limited to cancers, in many animal models.
RNA interference (RNAi) is a sequence-specific RNA degradation process that provides a relatively easy and direct way to knockdown, or silence, theoretically any gene containing the homologous sequence. In naturally occurring RNAi, a double-stranded RNA (dsRNA) is cleaved by an RNase III/helicase protein, Dicer, into small interfering RNA (siRNA) molecules, dsRNA of 19-27 nucleotides (nt) with 2-nt overhangs at the 3′ ends. Afterwards, the siRNAs are incorporated into a multicomponent-ribonuclease called RNA-induced-silencing-complex (RISC). One strand of siRNA remains associated with RISC to guide the complex towards a cognate RNA that has a sequence complementary to the guider ss-siRNA in RISC. This siRNA-directed endonuclease digests the RNA, resulting in truncation and inactivation of the targeted RNA. Recent studies have revealed the utility of chemically synthesized 21-27-nt siRNAs that exhibit RNAi effects in mammalian cells and have demonstrated that the thermodynamic stability of siRNA hybridization (at terminals or in the middle) plays a central role in determining the molecule's function. More detailed characteristics of RISC, siRNA molecules, and RNAi have been described in the scientific literature.
The utility of RNAi in down-regulation of mammalian cell gene expression has been shown successfully in the laboratory by utilizing either chemically synthesized siRNAs or endogenously expressed siRNA. The endogenous siRNA is first expressed as small hairpin RNAs (shRNAs) by an expression vector (plasmid or virus vector), and then processed by Dicer to become functional siRNAs.
Importantly, it is presently not possible to predict with any degree of confidence which of many possible candidate siRNA sequences potentially targeting a genomic sequence (e.g., oligonucleotides of about 16-30 base pairs) will in fact exhibit effective siRNA activity. Instead, individual, specific candidate siRNA polynucleotide or oligonucleotide sequences must be generated and tested to determine whether the intended interference with expression of a targeted gene has occurred. Accordingly, no routine method exists for designing an siRNA polynucleotide that is, with certainty, capable of specifically altering the expression of a given mRNA. Our process involves design of multiple siRNA sequences against a single gene and then testing of these sequences to validate their potency at silencing the selected gene as well as their selectivity (specificity for the target gene and not others).